Insecticidal compositions

ABSTRACT

An insecticidal composition comprising an enteric microorganism which is pathogenic to insects, and the enzyme chitinase. The insecticidal effect is increased by the concurrent contact of the insect with the chitinase. A typical microorganism is Bacillus thuringiensis.

. United States Patent [191 Smirnoff 51 Oct. 7, 1975 INSECTICIDALCOMPOSITIONS [75] Inventor: Wladimir A. Smirnoff, Ste. Foy,

Canada [73] Assignee: Canadian Patents and Development Limited, Ottawa,Canada [22] Filed: Dec. 22, 1972 [21] Appl. No.: 317,535

[52] U.S. Cl 424/93; 424/94 [51] A01N 15/00 [58] Field of Search 424/93,94

[56] References Cited UNITED STATES PATENTS 3,515,641 6/1970 Whitaker424/94 X OTHER PUBLICATIONS Smirnoff, W. A., Canadian Entomologist103(12): 1829-1831 Dec. (28) 1971, Effect of Chitinase on PrimaryExaminerShep K. Rose Attorney, Agent, or FirmAlan A. Thomson [5 7ABSTRACT An insecticidal composition comprising an enteric microorganismwhich is pathogenic to insects, and the enzyme chitinase. Theinsecticidal effect is increased by the concurrent contact of the insectwith the chitinase. A typical microorganism is Bacillus thuringiensis.

9 Claims, N0 Drawings INSECTICIDAL COMPOSITIONS This invention isdirected to insecticidal compositions based on enteromicroorganisms(including viruses) which are pathogenic to insects. Increasedinfectivity and insecticidal effect are obtained by the presence of theenzyme chitinase.

Recently the use of selected microorganism (bacteria, fungi and virus)preparations, which are natural enemies of the insects, has beendeveloped. These natural enemies of the insects usually are harmless toother forms of life, or at least are less harmful than many widely usedchemical insecticides. Preparations containing species of the genusBacillus and particularly Bacillus thuringiensis, are currently beingsprayed on a wide scale, particularly for the control of several speciesof Lepidoptera which attack agricultural crops. In many cases theinsecticidal action is slower and less widespread than would be desired.

Many of these microorganisms (including viruses) infect the alimentarycanal of the insect and an important controlling step in the rate andextent of insect mortality has been found to be the penetration of thegut tissues into the haemolymph. If this penetration could beaccelerated or facilitated the rate and extent of mortality might beexpected to be significantly increased.

In accordance with this invention, it has now been found that thecombination of or the concurrent use of the enzyme chitinase is able toincrease the insecticidal effectiveness of these enteric microorganisms(including viruses). The enzyme is combined with or applied with theinsecticidal microorganism preparation.

Chitinase is produced exocellularly by certain bacteria and fungi.Material can be obtained from various strains of Beauveria e.g., B.bassiana (Bals.) Vuill; Cordyceps; and Streptomyces inter alia and itcan be easily purified. Enzymatic activity in preparations is known toreach 2300 nephelometric units (units according to C. .leuniaux Chitineet Chitinolyse Masson, Paris, 1963 p. 27). In some of the testsdiscussed below a certified chitinase supplied by NutritionalBiochemicals Corp. U.S.A. was used. In the aerial spraying tests asemipurified material obtained from chicken entrails was used (about890-950 nephelometric units).

The amount of the chitinase needed is only a very small amount, usuallyless than about 1'7: by wt. of the composition. Even trace amounts havebeen found effective e.g., from about 3 to mg or more per lb. or gal. oftotal composition. The amount will also depend on the purity or activityof the enzyme preparation. Lab. Tests (as in Ex. 1 below) will readilyindicate an effective concentration.

The insecticidal microorganisms (including viruses) which may be usedinclude a number of varieties of Bacillus t/zuringiensis e.g., alesti,solto, endomycetis, dendrulimus, aizuwai, finitimus, galleriae,morrisoni, sub toxicus and itsserotype from 1 to 7. Chitinase can beused to facilitate the action of other entomopathogenic microorganismsthat act through the gutwall, particularly selected B. cereus varieties,B. popz'lliae, B. marital and other enterobacteriae. It can also be usedwith various entomopathogenic virus preparations particularly those ofnucleopolyhedrosis, cytoplasmic granulosis, and the non-included virusesand pox viruses of insects. The enzyme may also be used in conjunctionwith various species of entomopathogenic fungi or microsporidia.

A carrier or filler suitable for such insecticidal compositions isnormally used. Liquid carriers or diluents as would be used for sprayapplication include water, ethylene glycol, glycerol, higher molecularweight polyglycols, kerosene, various mineral oils, corn oil, whale oiland other organic liquids such as xylene, alcohols and ketones. Themixture can be in the form of an emulsion usually oil-in-water typeusing known emulsifying agents. Fillers or powders may be used to make adry composition suitable for dusting application. Dry powders of thebacteria (consisting mainly of spores or final whole culture) or viruscan be made by spray, vacuum or freeze-drying technique. Suitablepowders include talc, kaolin, bentonite and other clays, calciumcarbonate, zeolite, diatomaceous earth and alumina. In special casesbaits or vegetable powders such as bran or cereals can be used.

The insecticidal composition can also be applied by the aerosoltechnique using fluorinated hydrocarbon gases or other inert gases underpressure as propellant.

Chemical insecticides could be used in conjunction with themicrobial-enzyme system for an enhanced effect. Lesser amounts thannormally required of the chemical insecticide would suffice. Insectattractants may also be concurrently applied. Of course these chemicalsshould not significantly reduce the activity of the bacteria or virus.

The enzyme can be combined in small amounts with the microorganism(including virus) composition, or applied separately to the foliage orother locus frequented or consumed by the insects to be controlled.Sticking agents can be used to increase retention of the insecticide onthe foliage or locus.

The following Examples are illustrative:

EXAMPLE 1 The effect of various concentrations of the enzyme added totwo commercial preparations of B. tlzuringiensis and aqueous suspensionsof pure cultures of this bacillus obtained on nutrient agar, werestudied on larvae of C/wristoneura fumiferana reared on artificialmedium prepared by the method of Grisdale (December 1963 Rearing insectson artificial diet lnterium Res. Rept Insect Pathol. Res. Inst., SaultSte. Marie, Ont. Larvae were inoculated orally by placing a drop of thevarious suspensions tested on the artificial diet. Commercialpreparations of B. thuringiensis: Thuricide, trademark (internationalMinerals and Chemicals Corporation, Libertyville, Illinois, U.S.),serotype 5, var. Galleriae; and Dipel, (trademark) serotype 3, var.alesti, were used at a dosage of 2.8 billion international units. Theexperiment also included tests to determine the action of the Bacillusand chitinase separately.

Mixtures which gave significant results were then tested undertemperatures similar to those encountered in the field at the time(June); treatment with B. thuringiensis was used for control operations.The chitinase concentration was 10 mg. per lb. The experiments wererepeated under the following conditions: 12 hours at 22C, alternatingwith 12 hours at 16C and at a relative humidity of A second series ofexperiments with these concentrations were performed under constanttemperature of 16C and relative humidity. Each series with itsrespective control was repeated twice with 5 larvae and once with 20larvae. There was no larval mortality in control rearings at thespecified temperatures.

It was found that the activity of B. thuringiensis was considerablyincreased when chitinase was added to the preparation; larval mortalityprovoked by commercial preparations of B. thuringiensis was advanced byfrom 4 to 6 or more days for the various temperatures.

Pure cultures of these bacilli (cultured on nutrient agar) did notprovoke more than 50- 60% mortality, but in treatments using combinedbacilli and chitinase all the larvae succumbed from infection. Therapidity with which the insects died and the high degree of mortality,even at low temperatures, indicate that the action of b. thuringiensiswas directly increased by the presence of chitinase.

EXAMPLE 2 Applications of Bacillus thuringiensis by airplane or groundequipment have been used with little success in the control ofChoristoneura fun'ziferana Clemens. The reason is that the septicemiaenterotoxinosis, which provoke mortality of spruce budworm larvae, isinfluenccd by age and physiological condition of larvae, and byenvironmental conditions, especially temperature.

Infectivity tests carried out with B thuringiensis var. Galleriue(Serotype Thuricide HPC diluted l/10) on larvae of C. fumlj'erana rearedon artificial diet or on balsam fir (Abies balsamea Mill) foliage,revealed that the addition of 10 mg of chitinase to 500 g of Thuricide,increased the pathogenic action of the Bacillus and greatly increasedefficiency at low temperatures, ranging between 16- 18C. Larvalmortality in tests with chitinase occurred 5 to 6 days earlier than thatresulting from the action of the Bacillus alone and rapidly reached100%.

On the basis of these results an aerial spraying test was performed in astand of A. balsamea severely infected by Cfzmuferuna with Thuricide HPCalone, and with chitinase added. This experiment revealed that treatmentwith Bacillus plus chitinase arrested feeding of larvae and caused arapid and higher mortality than was the case with Thuricide alone. Also,more foliage was saved in the area treated with Thuricide plus chitinasethan in areas untreated or treated with Thuricide alone.

EXAMPLE 3 Experimental sprayings with commercial prepara tions of B.thuringiensis alone, and with chitinase, were carried out in balsam firstands severely infested by spruce budworm. Three l0O-acre plots wereestablished in these stands: Plot 1 was sprayed with Thuricide HPC pluschitinase, Plot 2 with Thuricide HPC alone, and Plot 3 served ascontrol. Spraying was done by a Steerman 'aircraft equipped withmicron-aire sprayers, which permitted an even and good coverage.

In Plot 1, 60.3% mortality occurred after 6 days, 78% mortality after 12days, and 88% mortality after 30 days following spraying. Forty-fivepercent of the pupae sample collected and brought to the laboratory foremergence died in rearing, making an over-all mortality of 94%. In Plot2 mortality reached 54% in 6 days, 72.4% in 10 days and 72.9% in 30 daysafter spraying. Over-all mortality reached 85.5%. In the control plot,no mortality occurred after 6 days, 42% mortality occurred after 10days, 47% after 30 days, and the over-all mortality was 65%.

To determine the protection of foliage resulting from spraying withThuricide, the 18 inch branch tips cut during the pupal check wereexamined in the laboratory. The results were: Plot 1: 24% of the budswere totally defoliated, 36% partially preserved, and 39 completelypreserved. Plot 2: Results were 64% totally defoliated, 31% partiallypreserved, and 4% completely preserved. Plot 3: 86% were totallydefoliated, 12% partially preserved, and 1% completely preserved. It wasalso established that B. thuringiensis survived for 30 days on foliage.

EXAMPLE 4 An extensive field trial was made on 10,000 acres ofbudworm-infested fir stands. Aerial spraying was carried out using thefollowing unit formula Thuricide HPC 0.5 gal Polyglycol liquid 0.5 galSticking agent 0.16 oz. Water l gal Chitinase 10 mg.

The chitinase used had an activity of about 950 nephelometric units.

The number of insects killed and the foliage saved were as good as thatobtainable by the commercial application of chemical insecticides andbetter than 0btainable from the application of Thuricide alone. Thisbiological insecticide would have a minimal effect on the ecology.

I claim:

1. An insecticidal composition comprising: (a) an enteric microorganismwhich is pathogenic to insects, and (b) the enzyme chitinase.

2. The composition of claim 1 wherein the pathogenic microorganism is anentomopathogenic enterobacteriae.

3. The composition of claim 2 wherein the enterobacteriae is one ofBacillus thuringiensis, Bacillus cereus, Bacillus popilliae and Bacillusmoritai.

4. The composition of claim 3 wherein the pathogenic bacteria is aBacillus thuringiensis preparation.

5. The composition of claim 1 wherein the chitinase is present in aneffective amount less than about l% by Wt.

6. The composition of claim 1 including a carrier or diluent.

7. The composition of claim 1 including a powdered filler.

8. A method of increasing the insecticidal effect of entericmicroorganisms pathogenic to insects, comprising contacting insects withthe microorganism concurrently with the enzyme chitinase.

9. The method of claim 8 wherein chitinase is combined with a Bacillusthuringiensis insecticide.

1. AN INSECTICIDAL COMPOSITION COMPRISING: (A) A ENTERIC MICROORGANISMWHICH IS PATHOGENIC TO INSECTS, AND (B) THE ENZYMECHITINASE.
 2. Thecomposition of claim 1 wherein the pathogenic microorganism is anentomopathogenic enterobacteriae.
 3. The composition of claim 2 whereinthe enterobacteriae is one of Bacillus thuringiensis, Bacillus cereus,Bacillus popilliae and Bacillus moritai.
 4. The composition of claim 3wherein the pathogenic bacteria is a Bacillus thuringiensis preparation.5. The composition of claim 1 wherein the chitinase is present in aneffective amount less than about 1% by wt.
 6. The composition of claim 1including a carrier or diluent.
 7. The composition of claim 1 includinga powdered filler.
 8. A method of increasing the insecticidal effect ofenteric microorganisms pathogenic to insects, comprising contactinginsects with the microorganism concurrently with the enzyme chitinase.9. The method of claim 8 wherein chitinase is combined with a Bacillusthuringiensis insecticide.